Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-06-22
2003-02-11
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S183000
Reexamination Certificate
active
06518040
ABSTRACT:
TECHNICAL FIELD
The present invention concerns, firstly, solid phase translation in general of mRNA to give a protein (polypeptide), encoded by the mRNA, and secondly, as a subaspect, region specific labelling of one or more predetermined regions (part sequences) of a polypeptide chain (protein) by solid phase translation of mRNA in vitro. The invention also encompasses novel region specific labelled proteins/polypeptides. The labelled polypeptides have their primary use in structural studies by NMR.
TECHNICAL BACKGROUND
The inventive method generalises an earlier mRNA-analogue (poly (U)) column translation method (Belitsina et al, 1975; Belitsina and Spirin, 1979) refined by Baranov et al., (1979), used to obtain ribosome in pre- and post-translocational states. Poly (U) as used in this earlier technique is not mRNA since it does not contain all the elements necessary for normal translation (stop codon, SD-sequence, (Shine-Delgarno sequence), ribosome binding site etc). Homopolymers of amino acids are not proteins. In the context of the invention homo polymers of amino acids are also excluded from the concept of polypeptides. In vitro translation has been described previously (e.g. Pavlov and Ehrenberg, 1996, and Ehrenberg et al, 1990).
NMR spectroscopy has over the past decade become a very powerful method to determine structures of small proteins in solution (Bax, 1989; Schwabe et al, 1990; Härd et al, 1990; Baumann et al, 1993; van Tilborg et al, 1995). NMR has the intrinsic limitation that, as the studied proteins get larger, there is a drastic reduction in the resolution of their NMR spectra (Bax, 1989). This drawback has been partially overcome by the application of various isotope labelling strategies (Muchmore et al, 1989; Ramesh et al, 1994). At the same time, there is still a pronounced upper limit around 30 kD for the determination of protein structures at high resolution using NMR spectroscopy (Bax, 1989).
One may identify three major types of isotope labelling strategies for NMR studies. The first is “uniform” labelling, where all the different amino acids in a polypeptide are labelled with, e.g.,
15
N,
14
C or
2
H isotopes. A combination of
15
N,
14
C or
2
H “uniform” labelling recently made it possible to determine the structure in solution of such a large molecular complex as the trp repressor in complex with operator DNA (Zhang et al, 1994).
A second strategy is “selective ” isotope labelling of proteins. This means that only a limited class of isotope labelled amino acids are built into the polypeptide, while the other amino acids are unlabelled. Selectively labelled proteins are obtained from over-producing bacterial strains, which grow in media where one or several types of amino acids are isotope labelled. This strategy has become very useful for structural analysis with NMR (Muchmore et al, 1989; Ramesh et al, 1994 and references therein).
A third strategy, which we denote “region specific” labelling, is more difficult to implement technically. This strategy means that labelled amino acids are incorporated only in one or more predetermined regions. One or more of all amino acid residues of a given peptide region may be labelled, while amino acids located outside the region may be unlabelled. We judge this, third strategy as the potentially most powerful way to extend the range of NMR-spectroscopy beyond the 30 kD limit to larger protein structures and complexes.
One way to obtain region specific labelling of proteins is by chemical polypeptide synthesis (Boutillon et al, 1995). At present, this method can only be used for small proteins.
OBJECTIVE OF THE INVENTION
A first objective is to provide an improved general method for in vitro translation enabling direct production of proteins in almost pure form.
A second objective is to provide a general method for region specific labelling of proteins based on in vitro translation as described e.g. by Pavlov and Ehrenberg, 1996, and Ehrenberg et al, 1990.
A third objective is to apply the solid support translation technology of the invention for implementing synthesis of region labelled proteins.
A fourth objective is to provide proteins that are isotope labelled at one or more predetermined regions.
The Invention
These objectives can be accomplished by a method that contemplates translation of real mRNAs stably linked to a solid phase to give real proteins polypeptides). These mRNAs contain in frame codons to be translated to an amino acid sequence. For procaryotes they also contain a Shine and Dalgarno sequence, a ribosornal binding site, an initiation codon and a termination codon, but one or several of these additional features may be deleted. For eucaryotes there is normally a cap structure at the 5′-end of the mRNA. One major advantage of this technique is that the ribosomes can be stalled at the stop codon that signals that the protein is full length (i.e., a terminating stop codon) as long as release factor is not included in the translation mixture. Subsequently, all components and factors necessary for translation can be rinsed off the solid phase in a simple way. After this step the solid phase linked mRNAs hold the ribosomes which hold the peptidyl-tRNAs that contain the protein of interest. Addition of the appropriate release factor (RF1 for UAA, UAG and RF2 for UAA or UGA) hydrolyses peptidyl-tRNA removing the protein of interest from the ribosomes that remain immobilized on the solid phase. Another rinsing step elutes the protein of interest together with catalytic amounts of RF1/2, making the final purification very simple.
Accordingly, the inventive method for production of proteins (polypeptides), is characterized in that mRNA encoding a protein of interest and bound to a solid phase is translated in vitro. In order to be able to obtain a highly purified form of the protein directly from the column translation is preferably done in two steps: first with a translation mixture containing all components for translation to a terminating, stop codon but devoid of the appropriate release factor activity—removal of the translation mixture—addition/introduction of the appropriate release factor activity.
A complete translation mixture is normally in the form of an aqueous buffer solution and allows for translation of the complete mRNA of interest and release of the so expressed protein from the tRNA—ribosome—mRNA complex. The mixture thus contains all ingredients necessary for translation, i.e. ribosomes, amino acids, amino acyl tRNA synthetases, tRNAs, initiation factors, elongation factors, energy giving system, buffering substances etc. See for instance the experimental part and references cited therein. The exact composition will depend on the origin of the various enzymes and factors utilized. For an
E. coli
origin, for instance, specific components, such as fmet-tRNA
fmet
, may have to be included.
By manipulating the translation mixture, translation may be paused and restarted at predetermined positions of the mRNA. The translation mixture may be added step-wise to the mRNA to be translated, such as a first portion (mixture) comprising ribosomes, initiation factors and for a procaryotic system fmet-tRNA
fmet
(for an eucaryotic system the initiator is met-tRNA
met
) (initiation mix) followed by a second portion (mixture) comprising elongation factors, amino acyl tRNA synthetases, transfer RNAs, amino acids, energy giving system etc (translation mix). In the preferred case the translation mixture is added stepwise with mixes varying in composition enabling pausing and restarting of translation at predetermined and well-defined positions.
A pause in translation may be achieved by eliminating from a mixture the amino acyl-tRNA activity hat reads the codon, where the translating ribosome shall stop. This may be achieved by removing the corresponding amino acid from the translation mixture, preferably in conjunction with a defective tRNA synthetase activity for that amino acid. Alternatively, the pausing may be at an internal stop codon in the mRNA. Readtbrough of the internal stop c
Ehrenberg Mans
Pavlov Michail
Achutamurthy Ponnathapu
Kerr Kathleen
Ronning, Jr. Royal N.
Ryan Stephen G.
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